CN111450321A

CN111450321A - Artificial skin substitute and scaffold-free self-assembly construction method and application thereof

Info

Publication number: CN111450321A
Application number: CN201910046704.5A
Authority: CN
Inventors: 王志刚; 陈虎; 姚咏明; 杨彩哲; 姜玉峰; 李冰; 李天民
Original assignee: Beijing Huafu Biotechnology Co ltd
Current assignee: Beijing Huafu Biotechnology Co ltd
Priority date: 2019-01-18
Filing date: 2019-01-18
Publication date: 2020-07-28

Abstract

The disclosure relates to an artificial skin substitute, a scaffold-free self-assembly construction method and application thereof. In particular, the present disclosure relates to an artificial skin substitute, a method of making the artificial skin substitute, and therapeutic uses of the aforementioned artificial skin substitute. The present disclosure relates to methods of making artificial skin substitutes that do not contain any scaffold components, such as synthetic scaffolds. Meanwhile, the artificial skin substitute disclosed by the invention does not relate to other substrates of animal origin, so that the immunogenicity of the product is reduced; and the use of human-derived materials significantly reduces or eliminates the costs associated with the production of animal-derived products and the risk of disease transmission resulting from the production of animal biologics.

Description

Artificial skin substitute and scaffold-free self-assembly construction method and application thereof

Technical Field

The present disclosure relates to the field of biomedical materials. In particular, it relates to an artificial skin substitute. More particularly, it relates to, inter alia, an artificial skin substitute and a method of scaffold-free self-assembly construction and use thereof.

Background

The skin tissue is the largest organ of the human body and is the first barrier for the human body to come into direct contact with the external environment. Skin defects resulting from severe skin wounds or burns are often difficult to repair by themselves to restore original morphology and function. Therefore, the skin substitute can be implanted through the operation, so that the healing speed can be increased, and the healing quality can be improved.

The artificial skin substitutes used in the prior art include Alloderm (human acellular lyophilized dermal cells), SureDerm (human acellular lyophilized dermal cells), OASIS Wound Matrix (porcine acellular lyophilized small intestinal submucosa cells), Biobrane (ultra-thin silicone as an epidermal mimic membrane, 3D nylon filament as a dermal analog containing type I collagen peptide), Integra (skin analog: bovine collagen and chondroitin-6-sulfate GAG; epidermal analog: silicone polymer polysiloxane), EpiDex (cultured keratinocytes from the root sheath outside the scalp), EPIBASE (cultured keratinocytes), Myskin (cultured keratinocytes), Decivorderm (autologous skin substitute), Pelnac (porcine tendon derived atedin type I, sponge layer), Dermagraf (Biodegradable human neonatal Matrix seeded with fibroblast), Cylite (autologous fibroblast (autologous reticulum microfiber (porcine tendon derived ateum type I), Biograft (autologous fibroblast-derived fibroblast-seeded with silicone), Biograft (Biograft-induced dermal graft induced by porcine tendon induced dermal growth), Biograft induced Wound healing induced by exogenous dermal growth factor induced by human fibroblast growth, Biograft-induced Wound healing process), Biograft induced by exogenous growth factor induced by human fibroblast growth factor induced by human growth, growth factor induced by human fibroblast growth, Biodermal growth factor induced by human growth, and induced by exogenous growth factor stimulation of human dermal growth factor induced by human growth factor, and induced by human growth factor, and induced by human growth factor, and induced by human growth factor induced by human growth, including human growth factor induced by human growth factor induced.

However, as mentioned above, although there are many types of artificial skin substitutes in the prior art, some types such as acellular, dermal or epidermal cells, no cells or only one cell type (e.g., fibroblasts or keratinocytes), and thus the cells secrete significantly less of the type and amount of matrix proteins available for wound healing, resulting in reduced efficacy.

At the same time, artificial skin substitutes such as Apligraf may promote wound healing of growth factors from fibroblasts and keratinocytes. Many composite skin grafts use animal-derived matrices, such as bovine collagen, for example, as a scaffold structure to construct the skin. For example, in the currently used artificial skin substitute, human dermis is formed by embedding human fibroblasts in bovine collagen, i.e., the artificial skin substitute is prepared using bovine collagen as a scaffold structure. Meanwhile, the prior art has adopted that fibroblasts of animal origin can be made to produce various extracellular matrices, including type I collagen, by prolonged culture under the condition of excessive fusion to embed their own cells to form dermis. However, since animal-derived collagen differs from the natural human skin extracellular matrix, the aforementioned products have only limited efficacy in healing wounds. In addition, these animal-derived products can cause immunogenicity and risk disease transmission. At the same time, the cost of producing these matrix proteins is high, and the differences between different production batches further increase the production costs.

For example, CN107349475B discloses an artificial tissue engineering skin with a nanofiber membrane and stem cells laminated one on top of the other. However, it is necessary to prepare an artificial skin substitute using adipose stem cells as a raw material, and during the process of preparing the artificial skin substitute, the activity of adipose stem cells is significantly reduced or killed.

CN108096624A discloses a method for preparing fibrinogen by using human blood, then forming a membrane from the fibrinogen, and further mixing the membrane with a skin growth assistant after the membrane is treated by a solution containing aprotinin to obtain a cytoskeleton. However, the aforementioned method uses a product directly derived from a human body (i.e., human blood) as a raw material, and thus the product obtained by the above method has a risk of disease transmission. At the same time, the above-described methods disclose materials that also contain skin growth aids, such as growth factors/vitamins, and the use of such ingredients also increases the production costs of the aforementioned methods.

US10149924B1 also discloses a directly usable biodegradable and biocompatible artificial skin substitute and a method for its preparation. However, the raw materials for preparation adopted by the chitosan/gelatin stent comprise chitosan/gelatin, and the chitosan/gelatin stent is not derived from human; meanwhile, the cells in the raw materials adopted in the examples are only one type of cells.

Therefore, it is of great importance to develop a new, low-cost, artificial skin substitute that is beneficial for improving the efficacy of wound healing.

Reference to the literature

1.Savoji,H.,et al.,Skin Tissue Substitutes and Biomaterial RiskAssessment and Testing.Front Bioeng Biotechnol,2018.6:p.86.

2.Bhardwaj,N.,D.Chouhan,and B.B.Mandal,Tissue Engineered Skin andWound Healing:Current Strategies and Future Directions.Curr Pharm Des,2017.23(24):p.3455-3482.

Disclosure of Invention

Problems to be solved by the invention

Based on the above problems in the prior art, the present disclosure provides an artificial skin substitute, and a scaffold-free self-assembly construction method and use thereof.

Means for solving the problems

In the technical scheme related to the present disclosure, the cells are cultured for a long time, so that the human cells used as raw materials for preparation can form the artificial skin substitute by relying on matrix proteins secreted by the cells, but not by other substances with exogenous scaffold structures.

In one embodiment, the present disclosure provides an artificial skin substitute comprising a first human-derived cell and a second human-derived cell as raw materials for preparation, and formed by stacking the first human-derived cell and the second human-derived cell; and, the preparation raw material does not contain a scaffold structure.

The artificial skin substitute according to the present disclosure, the first human-derived cell of the artificial skin substitute is selected from a fibroblast or a mesenchymal stem cell; optionally, the first human cell is selected from a fibroblast; preferably, the first human-derived cell is selected from human skin fibroblasts.

An artificial skin substitute according to the present disclosure, the second human cell of the artificial skin substitute being selected from keratinocytes; preferably, the second human-derived cell is selected from human epidermal keratinocytes.

In another embodiment, the present disclosure provides a method of preparing an artificial skin substitute comprising the steps of:

cell culture step: culturing the first human-derived cell and/or the second human-derived cell as a preparation raw material;

forming a dermal substitute without a scaffold cell source: culturing the first human-derived cell cultured in the cell culturing step as a cell to be cultured in the step of forming the scaffold-free cell-derived dermal substitute;

the forming step of the artificial skin substitute comprises the following steps: culturing the first human-derived cells obtained in the step of forming the scaffold-free cell-derived dermal substitute, and then co-culturing the second human-derived cells and the first human-derived cells to obtain an artificial skin substitute; and the number of the first and second electrodes,

the preparation raw materials do not contain a scaffold structure.

According to the preparation method of the artificial skin substitute provided by the present disclosure, the first human-derived cell is selected from a fibroblast or a mesenchymal stem cell; optionally, the first human cell is selected from a fibroblast; preferably, the first human-derived cell is selected from human skin fibroblasts.

According to the method for preparing the artificial skin substitute provided by the present disclosure, the second human-derived cell is selected from keratinocytes; preferably, the second human-derived cell is selected from human epidermal keratinocytes.

According to the method of preparing the artificial skin substitute provided by the present disclosure, the cells in the scaffold-free cell-derived dermal substitute are derived from fibroblasts.

According to the preparation method of the artificial skin substitute, in the step of culturing the first human-derived cells, a culture solution for culturing the fibroblasts is a DMEM culture solution; optionally, the DMEM culture solution is a high-glucose DMEM culture solution; preferably, the DMEM culture solution further contains fetal bovine serum; preferably, the weight volume ratio of the fetal calf serum in the DMEM culture solution is 2-20%; preferably, the weight volume ratio of the fetal bovine serum in the DMEM culture solution is 10%.

According to the preparation method of the artificial skin substitute provided by the present disclosure, in the culturing step of the second human-derived cells, the culture solution for culturing the keratinocytes is Derma L ife K culture solution or KGM culture solution.

According to the preparation method of the artificial skin substitute provided by the disclosure, in the step of forming the dermis substitute without the scaffold cell source, the initial inoculation concentration of the fibroblasts is 0.2-2X10⁶Per well; optionally, the culture solution of the fibroblast is 2-20% (weight/volume ratio)) Preferably, the content of the fetal bovine serum is 10%, preferably, the content of the ascorbic acid or the salt thereof is 100 mu g/m L, and preferably, the ascorbic acid or the salt thereof is selected from 2-phosphoric acid-L-ascorbic acid or 2-phosphoric acid-L-trisodium ascorbate.

According to the preparation method of the artificial skin substitute provided by the disclosure, the culture time of the fibroblasts is 2-6 weeks; preferably, the fibroblast cells are cultured for 4 weeks; optionally, the fibroblast cells are cultured at 37 deg.C in the presence of 5% CO₂Culturing under the conditions of (1).

The method for preparing an artificial skin substitute according to the present disclosure is characterized in that in the step of forming an artificial skin substitute, the step of forming a dermal substitute derived from the scaffold-free cells is performed, the cultured fibroblasts are collected, the culture solution is replaced, and after the keratinocytes are added, the culture solution is continuously cocultured with the fibroblasts.

The method for preparing the artificial skin substitute is provided according to the present disclosure, wherein the ratio of the fibroblasts to the epidermal keratinocytes is selected from 1:1 to 1: 4; optionally, the ratio of the fibroblasts to the epidermal keratinocytes is 1: 2.

According to the preparation method of the artificial skin substitute, the replaced culture solution is a reduced FAD culture solution, optionally, the reduced FAD culture solution is a culture solution containing fetal bovine serum and ascorbic acid or a salt thereof, preferably, the weight-volume ratio of the fetal bovine serum in the reduced FAD culture solution is 2-20%, preferably 10%, preferably, the content of the ascorbic acid or the salt thereof in the reduced FAD culture solution is 10-200 μ g/m L, preferably 100 μ g/m L, and preferably, the ascorbic acid or the salt thereof is selected from 2-phosphoric acid-L-ascorbic acid or 2-phosphoric acid-L-trisodium ascorbate.

According to the preparation method of the artificial skin substitute provided by the disclosure, the initial inoculation concentration of the keratinocyte is 0.25-2 × 10⁵Per well.

According to the preparation method of the artificial skin substitute, the fibroblasts and the keratinocytes are subjected to submerged co-culture to obtain an epithelial cell co-culture, and then the epithelial cell co-culture is obtained to obtain the artificial skin substitute; optionally, the obtaining is an air stripping outer loop flow obtaining.

In another embodiment, the present disclosure also provides an artificial skin substitute, which is obtained by the aforementioned preparation method.

In another embodiment, the present disclosure also provides a use of an artificial skin substitute for the preparation of a medical material for the treatment of a skin defect; optionally, the skin defect is a skin burn.

In another embodiment, the present disclosure also provides a method of treating a skin injury, characterized by applying the artificial skin substitute of the present disclosure to a skin defect; optionally, the skin defect is a skin burn.

ADVANTAGEOUS EFFECTS OF INVENTION

In one embodiment, the present disclosure is directed to a method of making an artificial skin substitute that is prepared from starting materials that do not contain any scaffold-structured components. Illustratively, the preparation method and the product prepared by the foregoing method do not comprise materials or matrices of animal origin or of artificial synthesis.

In one embodiment, the present disclosure uses human fibroblasts to produce dermis and keratinocytes to produce epidermis, without involving other animal-derived substrates, thus reducing the immunogenicity of the product; at the same time, the use of human-derived materials can significantly reduce or eliminate the costs associated with the preparation of animal-derived products and the risk of disease transmission resulting from the preparation of animal biologics.

In another embodiment, the present disclosure uses starting materials that are free of synthetic materials, thereby reducing the cost of preparing the relevant synthetic materials and reducing the risk of strong immunogenicity and toxicity that may be present.

In one embodiment, the present disclosure improves graft uptake and enhances wound healing efficacy by allowing fibroblasts to secrete extracellular matrix that can mimic the in vivo environment of human skin, thereby utilizing human fibroblasts to form the dermis.

In another embodiment, the present disclosure is applied to an artificial skin substitute by controlling the ratio and/or the order of addition of fibroblasts (fibroplasts) and keratinocytes (keratinocytes) in the culture broth during the preparation of the artificial skin substitute by cell co-culture, thereby allowing the cultured cells to form a skin-like structure.

In one embodiment, the present disclosure may adjust the thickness of the artificial skin substitute prepared by extending the culture time of fibroblasts, adjusting the composition of the culture solution of fibroblasts, and periodically spreading the fibroblasts on the epidermal layer.

Drawings

FIG. 1 is a schematic diagram showing morphological observation of fibroblast cultures under an optical microscope.

FIG. 2 is a schematic view showing morphological observation of keratinocytes under an optical microscope.

Fig. 3 shows a schematic diagram of a transwell in a deep-well plate.

Fig. 4 shows a schematic diagram of a mature 3D skin structure.

FIG. 5 is a graph showing the staining results of hematoxylin-eosin (H & E) for the artificial skin substitute.

FIG. 6 is a graph showing the effect of fibroblast seeding density on the thickness of an artificial skin substitute. The density of fibroblasts was 0.2 ten thousand cells/well (a), 0.6 ten thousand cells/well (B) or 200 ten thousand cells/well (C), and used for the construction results of the artificial skin substitute.

Detailed Description

Definition of

In the claims and/or the description of the present disclosure, the words "a" or "an" or "the" may mean "one", but may also mean "one or more", "at least one", and "one or more than one".

As used in the claims and specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Also, the terms "comprising," "having," "including," or "containing" are intended to be inclusive and mean that there may be additional, unrecited elements or method steps.

Throughout this specification, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless expressly indicated to be merely an alternative or a mutual exclusion between alternatives.

By "animal source" in this disclosure is meant a source of non-Homo sapiens (Homo sepiens). Exemplarily, the "animal-derived cell" in the present disclosure refers to a cell derived from non-Homo sapiens (Homo sepiens), for example, a cell derived from Bovine (Bovine).

The "scaffold structure" in the present disclosure refers to a cell matrix of animal cell origin or an artificially synthesized material. Illustratively, the scaffold structure may be collagen of animal origin or a synthetic nanofiber membrane.

The "Fibroblast (Fibroblast)" in the present disclosure is also called Fibroblast, is a main cellular component of loose connective tissue, and belongs to terminally differentiated cells, which produce proteins such as collagen and are differentiated from mesenchymal cells (mesenchymeal cells) at the embryonic stage. Fibroblasts are large and clear in outline, mostly have a protruded spindle-shaped or star-shaped flat structure, the nucleus of the fibroblast is in a regular oval shape, and the kernel is large and obvious.

The "Mesenchymal Stem Cell (MSC)" in the present disclosure is a multipotent stem cell, which is an adult stem cell that exists in various tissues (e.g., bone marrow, umbilical cord blood and umbilical cord tissue, placenta tissue, adipose tissue, etc.), has a multipotent differentiation potential, and is not a hematopoietic stem cell. The stem cells have the potential of differentiating into various mesenchymal series cells (such as osteogenic cells, chondrogenic cells, adipogenic cells and the like) or non-mesenchymal series cells, and have unique cytokine secretion functions.

The "keratinocytes (keratinocytes)" in the present disclosure are the major constituent cells of the epidermis, accounting for more than 80% of the epidermal cells, and produce keratin during differentiation. The differentiation stage and characteristics of the traditional Chinese medicine composition can be divided into five layers, namely a basal layer, a spinous layer, a granular layer, a transparent layer and a cuticle from inside to outside.

The "hematoxylin-eosin (H & E) staining" in this disclosure is a commonly used method of cell staining. The cell nucleus is stained bright blue by hematoxylin, the cartilage matrix and calcium salt particles are dark blue, and the mucus is gray blue. The cytoplasm is stained by eosin in a shade of pink to pink, and the eosinophilic granules in the cytoplasm are bright red with strong light reflection. Collagen fiber is light pink, elastic fiber is bright pink, erythrocyte is orange red, and protein liquid is pink.

The "DMEM medium (Dulbecco's modified Eagle's medium)" in this disclosure is a medium containing various amino acids and glucose, which is developed on the basis of MEM medium, and is divided into a high-sugar DMEM medium (glucose content less than 4500 mg/L) and a low-sugar DMEM medium (glucose content less than 1000 mg/L) while increasing the amounts of various components compared to MEM.

Detailed description of the preferred embodiments

Hematoxylin-eosin (H)&E) Dyeing experiment

It will be appreciated by those skilled in the art that H & E staining is a widely used staining method in histology. At the same time, many variations and protocols for H & E staining have been developed in the prior art, and the disclosure is not limited to any particular variables, protocols, systems, etc., insofar as the disclosure can be applied. A general H & E system can be defined to include four core staining reagents: hematoxylin, differentiating agents, bluing agents and eosin, as well as the cleaning agents and solvents required for dewaxing, rehydration, dehydration and clearing (clearing).

A typical H & E staining protocol generally comprises the following steps: (i) dewaxing (using xylene or xylene substitutes); (ii) rehydration (using alcohols); (iii) washing with water; (iv) hematoxylin; (v) washing with water; (vi) a differentiating agent; (vii) washing with water; (viii) bluing; (ix) washing with water; (x) An alcohol; (xi) Eosin; (xii) Dehydration (using alcohols); and (xiii) (using xylene or xylene substitutes) transparentization (Sheenan, D.C. and Hrapchak, B.B.: Theory and Practice of Histotechnology 2nd ed. Columbus, OH, Battelle Press, 1980).

In one embodiment, the present disclosure is applied to an artificial skin substitute by controlling the ratio and/or the order of addition of fibroblasts (fibroplasts) and keratinocytes (keratinocytes) in the culture broth during the preparation of the artificial skin substitute by cell co-culture, thereby allowing the cultured cells to form a skin-like structure.

In one embodiment, the aforementioned ratio of fibroblasts to keratinocytes may be selected from 1:1 to 1: 4.

In a preferred embodiment, the ratio of the aforementioned fibroblasts to keratinocytes is 1: 2.

In another embodiment, fibroblasts are added first, followed by keratinocytes during cell co-culture. In this way, fibroblasts can be more easily controlled so that they do not overgrow and keratinocytes can be prevented from covering the entire surface of the cell culture broth. Thereby enabling the cultured cells to form a skin-like structure for use as an artificial skin substitute.

In one embodiment, the present disclosure may increase the thickness of the artificial skin substitute produced by extending the time of culturing fibroblasts, adjusting the composition of the culture solution of fibroblasts, and periodically plating the fibroblasts on the epidermal layer.

In an alternative embodiment, the culture time of the fiber cells may be selected from 2-8 weeks.

In a preferred embodiment, the fiber cells are cultured for 4 weeks.

In an alternative embodiment, ascorbic acid is added to the culture broth from which the fibroblasts are cultured to increase the thickness of the artificial skin substitute produced. Applicants have found that artificial skin substitutes of increased thickness can be prepared by adding ascorbic acid, which in turn accelerates the synthesis and secretion of collagen by human fibroblasts.

In a preferred embodiment, the ascorbic acid is added in an amount of 10-100. mu.g/ml.

In a preferred embodiment, fibroblasts are plated on the epidermal layer every 5-10 days.

Examples

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

All reagents used in the examples were commercially available unless otherwise noted.

Example 1: method for culturing fibroblast

NHDF (Normal human dermal fibroblasts ) was added to 2% (w/v) fetal calf serum (FCS, Invitrogen) in high glucose DMEM medium (L naza) at 37 deg.C with 5% CO₂Culturing under the conditions of (1). When the cells are cultured until the culture plate is fully paved by 70% -90%, culturing the cultured fibroblasts for later use.

Example 2: method for culturing fibroblast

Example 2 the culture method was similar to that of example 1, except that 10% (weight/volume) fetal bovine serum (Invitrogen) was added to the high-glucose DMEM culture solution (L onza) (instead of 2% fetal bovine serum in example 1), and the culture conditions were otherwise the same.

Practice ofExample 3: method for culturing fibroblast

Example 3 the culture method was similar to that of example 1, except that 20% (weight/volume ratio) fetal bovine serum (Invitrogen) was added to the high-glucose DMEM culture solution (L onza) (instead of 2% fetal bovine serum in example 1), and the culture conditions were otherwise the same.

The culture results of fibroblasts cultured in examples 1 to 3 were similar. Exemplary culture results of fibroblasts cultured in example 2 are shown in fig. 1.

Example 4: method for culturing mesenchymal stem cells

The cell culture conditions of example 4 were the same as in example 1. That is, example 4 the same culture method as example 1 was used to culture mesenchymal stem cells.

Example 5: method for culturing mesenchymal stem cells

The cell culture conditions of example 5 were the same as those of example 2. That is, example 5 the same culture method as example 2 was used to culture mesenchymal stem cells.

Example 6: method for culturing mesenchymal stem cells

The cell culture conditions of example 6 were the same as those of example 3. That is, example 6 the same culture method as example 3 was used to culture mesenchymal stem cells.

Example 7: method for culturing keratinocyte

NHEK (Normal human epidermal keratinocytes) were cultured in Derma L if K broth (L ifeline). The cultured fibroblasts were cultured until they were confluent with 70% -90% plates.

Example 8: method for culturing keratinocyte

Example 8 the culture method was similar to that of example 7, except that Derma L ife K broth (L ifeline) was replaced with KGM broth (L onza), and the culture conditions were otherwise the same.

The culture results of keratinocytes cultured in examples 7-8 were similar. Exemplary culture results of keratinocytes cultured in example 7 are shown in FIG. 2.

Example 9: formation of a dermal substitute of stentless cell origin

0.2-2X10 obtained by culturing in example 1-3⁶NHDF cells were seeded into translucent 6-well deep-well cell culture plates (transwell, Corning) at 37 ℃ in 5% CO₂Under the cell culture conditions of (3), for 2 to 6 weeks.

Among them, the cell culture solution of NHDF is DMEM (L onza) containing 2-20% fetal calf serum (FCS, Invitrogen) and 10-200. mu.g/m L2-phosphate-L-ascorbic acid (Sigma).

The results of culturing keratinocytes cultured in example 9 are shown in FIG. 3.

Example 10: formation of artificial skin substitute

The NHDF cells obtained by the culture in example 9 were collected, and the culture medium of the cells was replaced, and then the culture was continued in the cell culture well for 2 to 8 weeks.

Wherein the culture medium after replacement is reduced FAD (rFAD), the rFAD culture medium contains 2-20% fetal calf serum (FCS, Invitrogen), 10-200 μ g/m L2-phosphoric acid L-ascorbic acid trisodium salt (Sigma), 0.4-20 μ g/m L hydrocortisone (Sigma), and1 × 10^-10M cholera toxin (Sigma).

After NHDF cells were cultured in the rFAD culture medium for 2 to 8 weeks, 0.25 to 2 × 10 was added to each cell culture well⁵NHEK cells obtained by culturing according to examples 7-8. Optionally, the content ratio of the NHDF cells to the NHEK cells is 1:1-1: 4. In this example, the applicant employed the content ratio of the aforementioned NHDF cells to NHEK cells to be 1: 2.

The NHDF cells and NHEK cells were subjected to submerged co-culture, and after 2-7 days of growth, the co-culture was subjected to air-lift-induced external circulation (air-lifted) and the culture medium was changed every other day.

Example 10 the 3D skin structure of the cultured stentless artificial skin substitute is shown in fig. 4.

Example 11: hematoxylin-eosin (A), (B)H&E) Dyeing experiment

The non-scaffolding artificial skin substitute obtained by the culture in example 10 was stored in a 10% neutral buffered formalin solution, and then stained with hematoxylin-eosin (H & E).

From the staining results, it was observed that the artificial skin substitute obtained by the aforementioned culture had a structure of epidermis and dermis. Since the raw materials for preparing the artificial skin substitute are all of human origin, the product prepared in example 10 can be used as a stentless artificial skin substitute for human body according to the dyeing result.

Example 12: thickness determination of stentless artificial skin substitutes

For the stentless artificial skin substitute obtained in example 11, an image of the skin substitute was captured at 20 times magnification and its thickness was calculated using a reference scale.

The results of the experiment are shown in FIG. 6. As can be seen from fig. 6, the seeding density of the original fibroblasts had a significant effect on the thickness of the resulting stentless artificial skin substitute. In FIG. 6, the density of fibroblasts is 0.2 ten thousand cells/well (A), 0.6 ten thousand cells/well (B), or 200 ten thousand cells/well (C), respectively

When the initial fibroblast seeding density is 0.2-2x10⁶At each cell, the thickness of the finally obtained scaffold-free artificial skin substitute is 20-180 μm.

Example 14: thickness influencing factor of stentless artificial skin substitute

It should be noted that the variation in thickness of the artificial skin substitute, although important, is only a part of the consideration in practical applications. In particular, there is no report of a direct and inevitable correlation between the thickness and efficacy of artificial skin substitutes, it being understood that thick skin is easily transplanted to treat wounds, but implanting more cells increases production costs. In fact, in many cases, the patient need only use fused cells cultured on a cell plate for wound healing.

Applicants have found that the thickness of the artificial skin substitute can be increased by the following embodiments.

In one embodiment, the fibroblast cells may be cultured for a correspondingly extended period of time. In the technical scheme of the disclosure, the suitable culture time of the fibroblasts is 2-8 weeks. Therefore, by extending the culture time of fibroblasts, the thickness of the cultured scaffolds-free artificial skin substitute is obviously increased. In a preferred embodiment, the fibroblasts are cultured for a period of 4 to 8 weeks.

In another embodiment, ascorbic acid may be added to the culture broth used to culture fibroblasts in the presently disclosed embodiments in an amount of 10-200 μ g/m L. thus, the thickness of the stentless artificial skin substitute obtained from the culture may be significantly increased by adding a higher concentration of ascorbic acid, in a preferred embodiment, ascorbic acid is added in an amount of 100 μ g/m L.

The above examples of the present disclosure are merely examples provided for clearly illustrating the present disclosure and are not intended to limit the embodiments of the present disclosure. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present disclosure should be included in the protection scope of the claims of the present disclosure.

Claims

1. An artificial skin substitute, characterized in that the artificial skin substitute comprises a first human-derived cell and a second human-derived cell as raw materials for preparation, and is formed by superposing the first human-derived cell and the second human-derived cell; and, the preparation raw material does not contain a scaffold structure.

2. The artificial skin substitute according to claim 1, wherein the first human-derived cell of the artificial skin substitute is selected from a fibroblast or a mesenchymal stem cell; optionally, the first human cell is selected from a fibroblast; preferably, the first human-derived cell is selected from human skin fibroblasts.

3. The artificial skin substitute according to claim 1 or 2, wherein the second human-derived cells of the artificial skin substitute are selected from keratinocytes; preferably, the second human-derived cell is selected from human epidermal keratinocytes.

4. A preparation method of an artificial skin substitute is characterized by comprising the following steps:

the preparation raw materials do not contain a scaffold structure.

5. The method of claim 4, wherein the first human-derived cell is selected from a fibroblast cell or a mesenchymal stem cell; optionally, the first human cell is selected from a fibroblast; preferably, the first human-derived cell is selected from human skin fibroblasts.

6. The method according to claim 4 or 5, wherein the second human-derived cell is selected from the group consisting of a keratinocyte; preferably, the second human-derived cell is selected from human epidermal keratinocytes.

7. The method of claim 4, wherein the cells in the scaffold-free cell-derived dermal substitute are derived from fibroblasts.

8. The method according to claim 5, wherein in the step of culturing the first human-derived cells, a culture solution for culturing the fibroblasts is a DMEM culture solution; optionally, the DMEM culture solution is a high-glucose DMEM culture solution; preferably, the DMEM culture solution further contains fetal bovine serum; preferably, the weight volume ratio of the fetal calf serum in the DMEM culture solution is 2-20%; preferably, the weight volume ratio of the fetal bovine serum in the DMEM culture solution is 10%.

9. The method according to claim 6, wherein a culture medium for culturing the keratinocytes in the step of culturing the second human-derived cells is Derma L ife K culture medium or KGM culture medium.

10. The method of claim 7, wherein the fibroblast cells are initially seeded at a concentration of 0.2-2X10 during the step of forming the scaffold-free dermal substitute⁶The culture solution of the fibroblast is DMEM culture solution containing 2-20% (weight to volume) of fetal bovine serum and 10-200 mu g/m L of ascorbic acid or a salt thereof, preferably the fetal bovine serum is 10%, preferably the ascorbic acid or the salt thereof is 100 mu g/m L, and preferably the ascorbic acid or the salt thereof is selected from 2-phosphoric acid-L-ascorbic acid or 2-phosphoric acid-L-trisodium ascorbate.

11. The method according to claim 10, wherein the fibroblast is cultured for 2 to 6 weeks; preferably, the fibroblast cells are cultured for 4 weeks; optionally, the fibroblast is cultured at 37 deg.C under 5%CO₂Culturing under the conditions of (1).

12. The method according to claim 4, wherein in the step of forming the artificial skin substitute, the step of forming the dermal substitute derived from the scaffold-free cells comprises collecting fibroblasts obtained by culturing, replacing the culture medium, adding the keratinocytes, and then continuously co-culturing the fibroblasts.

13. The method of claim 12, wherein the ratio of the fibroblasts to the epidermal keratinocytes is selected from 1:1 to 1: 4; optionally, the ratio of the fibroblasts to the epidermal keratinocytes is 1: 2.

14. The preparation method of claim 12 or 13, wherein the replaced culture solution is a reduced FAD culture solution, optionally the reduced FAD culture solution is a culture solution containing fetal calf serum and ascorbic acid or a salt thereof, preferably the fetal calf serum weight volume ratio in the reduced FAD culture solution is 2-20%, preferably 10%, preferably the ascorbic acid or a salt thereof in the reduced FAD culture solution is 10-200 μ g/m L, preferably 100 μ g/m L, preferably the ascorbic acid or a salt thereof is selected from 2-phosphate-L-ascorbic acid or 2-phosphate-L-ascorbic acid trisodium salt.

15. The method of claim 13, wherein the keratinocyte is initially seeded at a concentration of 0.25-2 × 10⁵Per well.

16. The method of claim 12, wherein the fibroblasts and keratinocytes are subjected to submerged co-culture to obtain an epithelial cell co-culture, and the epithelial cell co-culture is further obtained to obtain the artificial skin substitute; optionally, the obtaining is an air stripping outer loop flow obtaining.

17. An artificial skin substitute obtained by the production method according to any one of claims 4 to 16.

18. Use of an artificial skin substitute according to any one of claims 1 to 3 or claim 17 for the preparation of a medical material for the treatment of a skin defect; optionally, the skin defect is a skin burn.

19. A method of treating a skin lesion, characterized in that an artificial skin substitute according to any one of claims 1 to 3 or claim 17 is applied to a skin defect; optionally, the skin defect is a skin burn.